A vaccine to combat malaria is a highly desirable public health tool to reduce morbidity and mortality in African children. In order to achieve this goal it will be essential to gain a detailed understanding of the impact of malaria on the generation and maintenance of immunological memory, the basis of all vaccines. Our goal is to gain an understanding of the generation, maintenance and activation of immunological memory in response to natural malaria infection, focusing on B cells and the function of Pf-specific antibodies. We determined the acquisition of both Pf-specific memory B cells (MBCs) and Pf-specific Abs using a proteome chip containing approximately 25% of the Pf proteome in a cohort of children and young adults in Kambila, Mali exposed to intense, seasonal Pf transmission. We documented that both the long-lived Pf-specific Abs and MBCs increased slowly, year by year, in a step-wise fashion. This year we established a collaboration with Drs. Turner and Lavstsen (U. Copenhagen) and Dr. Smith (Seattle Biomedical Research Institute) to characterize the acquisition of antibodies specific for the Pf variant antigen, Pf erythrocyte membrane protein 1 (PfEMP1). PfEMP1s are expressed on the surface of infected red blood cells (iRBCs) and play an essential role in sequestering the iRBC in tissue microvasculature, thus avoiding clearance in the spleen. The var genes that encode PfEMP1 are highly polymorphic and each parasite clone expresses one var at a time but can readily switch to the expression of an antigenically unrelated var under immune pressure. PfEMP1s can be classified into three groups, A, B and C. Current evidence suggest that antibodies to Var A are acquired first in children in endemic areas and that var As are involved in the most severe forms of malaria. Thus, understanding the acquisition of PfEMP1s may aid in the development of vaccines that present severe malaria. We will screen serum from our cohort for antibodies to the var A, B and C PfEMP1s using proteins produced by the Copenhagen group and establish the order of acquisition of specific antibodies and their longevity. Although classical antigen-specific MBCs were generated in response to malaria infections we also observed a large increase in the number of atypical FcRL4+ MBCs. Atypical FcRL4+ MBCs appear to be functionally impaired and activated only poorly through their B cell receptor (BCR). We subsequently showed FcRL4+ atypical MBC were increased in Peruvian adults exposed to low Pf transmission, but not to the extent observed in Malian adults (Peru mean 5.4%; Mali mean 13.1%) suggesting a correlation between Pf exposure and the generation of atypical FcRL4+ MBCs. We recently observed that children in rural Kenya exposed to ongoing Pf transmission had larger numbers of atypical FcRL4+ MBCs as compared to age-matched controls living in similar conditions but in an area where Pf transmission ceased five years earlier, suggesting that Pf drives the generation or maintenance of atypical FcRL4+ MBCs. To further understand the specificity of atypical MBCs we carried out studies to determine the antibody repertoire of conventional FcRL4- and atypical FcRL4+ MBCs. We cloned VH and VL genes from single B cell from atypical FcRL4+ and conventional FcRL4- MBCs from Malian children and adults and thus far have acquired approximately 200 sequences from both Malian children and adults. We will also carry out deep sequencing of plasma blast that are greatly expanded in the peripheral blood approximately seven days after a case of malaria and are enriched in Pf-specific cells. We have recently expanded our analysis of the antibody repertoire to describe the structure of the entire human antibody repertoire in response to malaria in collaboration with Dr. Ning Jiang, University of Texas. To do so we will use high-throughput-long read sequencing to characterize the expressed antibody repertoire in children and adults living in malaria-endemic Mali and the impact of malaria on this repertoire. We will also clone VH and VL from individual B cells and express those as antibodies to determine which sequences contribute to the Pf-specific immune response. To understand how Pf infection alters the acquisition of specific antibodies we have explored the interactions between Pf and the human host. We carried out an extensive yeast two hybrid screen between 150 Pf proteins anticipated to be exposed to the human immune system and 12,000 human open reading frames. We identified several potential interactions and have made significant progress on one. We showed that the 33kD fragment of Pf merozoite surface protein 1 (MSP133), an abundant merozoite surface protein that is shed during red blood cell invasion, binds to S100P in a calcium dependent fashion, a member of S100 family, several of which are pro-inflammatory, damage associated molecular pattern proteins. Our recent studies showed that all known S100 family members bind to MSP1-33 but differ in their metal ion requirements. MSP133 binds to S100P with high-affinity and blocks S100P-induced NFkappaB activation in monocytes and chemotaxis and superoxide production in neutrophiles. Remarkably, S100P binds to both dimorphic alleles of MSP1, estimated to have diverged over 27 million years ago, suggesting an ancient conserved relationship between these parasite and host proteins. We postulate that MSP133 is released by Pf merozoites to attenuate potentially damaging inflammatory responses for the benefit of both the parasite and the host. We are currently attempting to determine the structure of S100 bound to MSP1-33 to determine if this binding site corresponds to the binding site of other S100s for their receptors. From these studies we hope to gain insight into the possible mechanism by which MSP133 interferes with S100 function. S100B is highly expressed in brain where it has been shown to have an anti-inflammatory role. We are currently testing selective S100B inhibitors for their effects on cerebral malaria in a mouse model. Over the last year we also explored the relationship between Pf infections and autoimmune disease. In collaboration with Dr. Silvia Bolland, we provided evidence that a genetic susceptibility to systemic lupus erythematosus (SLE) protects against cerebral malaria in a mouse model. Protection appears to be by immune mechanisms that allow SLE-prone mice to better control their overall inflammatory responses to parasite infections. We are exploring the possibility that anti-inflammatory therapy may treat cerebral malaria. In addition, in collaboration with Dr. Inaki Sanz (Emory University) we are characterizing our Malian cohort for autoantibodies associated with human SLE. VH4-34-containing antibodies are auto-antibodies and can be identified using the VH4-34-specific monoclonal antibody 9G4. 9G4+ memory B cells and serum antibodies are prevalent in SLE but not in healthy controls. We have recently determined that in malaria-exposed Malians 9G4+ antibodies rise after clinical malaria but are controlled and rapidly disappear and do not appear in the memory responses. These observations suggest a special but distinct regulation of 9G4+ B cells and antibodies in SLE and in malaria. Our studies of severe cerebral malaria in mice has led us to better characterize the sequence of events that contribute to the disease. To do so we have established a collaboration with Dr. Dorian McGavern to use two photon, intravital imaging to view the cellular events that occur following infection with a mouse Plasmodium that results in cerebral disease using fluorescently labeled parasites, T cell, mononuclear cells and markers for vascular permeability.